In Project 2, primary cortical neuronal, astroglial and mixed neuronal-astroglial cultures will be used to investigate whether proteins that show increased expression in an in vivo model of ischemic preconditioning and tolerance (Project 1) are cytoprotective in vitro. The hypothesis is that proteins showing increased expression in animal models of ischemic tolerance in vivo (Project 1) can be screened using cell culture models of ischemia and tolerance in vitro (Project 2) to identify proteins that will, in turn, protect neurons, astroglia or both from ischemia in vivo (Project 3). The specific aims are to [1] determine which candidate cytoprotective proteins show increased expression in a cerebral cortical neuronal, astroglial or mixed neuronal-astroglial cell-culture model of tolerance to oxygen and glucose deprivation (OGD); [2] investigate which candidate cytoprotective proteins protect cultured cortical neurons, astroglia or both from otherwise lethal OGD, by determining the effect on cell viability of increasing their expression or (for secreted proteins) their extracellular concentration; [3] investigate which candidate cytoprotective proteins protect cultured cortical neurons, astroglia or both from otherwise lethal OGD, by determining the effect on cell viability of reducing their expression or inhibiting their function; and [4] investigate the mechanism of action of candidate cytoprotective proteins identified in Project I and confirmed in Specific Aims 1-3 of Project 2. The most promising candidate cytoprotective proteins that emerge from Project 2 will then be tested for protective effects in vivo in Project 3. The methods used will include primary culture of rat cerebral cortical neurons and astroglia; in vitro models of cerebral ischemia, preconditioning and tolerance; cell viability assays; Western blotting; immunocytochemistry; transfection with adeno-associated virus vectors; and transgenic and gene knockout mice. The broad, long-term objective is to identify endogenous protective mechanisms in ischemia and to exploit these in the treatment of stroke.